The proposed experiments will test the hypothesis: The separation of leukocytes by their intrinsic properties using Dean Flow Fractionation will enable analyses of innate and adaptive immune responses in microliter quantities of whole blood. Acute and chronic inflammatory responses are integral to the clinical expression of many diseases, yet routine clinical assessment of immune function is based solely on leukocyte count. This is typically measured with a complete blood count and leukocyte differential count. In current clinical practice, critical clinical decisions regarding immune modulation are made based on the count of leukocytes and leukocyte subclasses without assessment of leukocyte function. In addition, the measurement of a peripheral venous complete blood count uses 3-5 mL. Based on the significant blood volume required for the complete blood count and other routine laboratory testing, patients can develop iatrogenic anemia. There is a critical need to develop assays that enable assessment of immune function and require significantly smaller volumes of blood. In preliminary results, we have developed an inertial microfluidic approach to selectively separate diverse cellular targets that is based on non-labeled, cell type specific physical features. From peripheral venous blood, this new approach can separate leukocytes from erythrocytes and platelets, leukocyte subclasses and distinguish activated neutrophils from unactivated cells by tandem iso-dielectric separation. This multi-functional, extracorporeal separation system is high throughput and capable of preparatory separation of leukocytes prior to coupled analyses of systemic and cellular immune function. To address our hypothesis, two specific aims are proposed: 1. Design microliter scale preparatory inertial microfluidic separation of leukocytes in peripheral blood; 2. Determine microliter scale assays of systemic and cell-based immune function. Recent advances in high-throughput microfluidic engineering enable this project, paving the way for a new approach to complex immune monitoring.